Class 18 myosins are most closely related to conventional class 2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, arguing that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the second longest coiled-coil of any myosin outside of the class 2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any conditions tested. Instead, M18A formed a 65 nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments, and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the myriad cellular and developmental roles attributed to NM2, our results have far reaching biological implications. Class-18A myosins are a poorly understood subclass of myosins with a domain architecture similar to that of class II myosins. In contrast to class II myosins though, myosin 18A has no ATPase activity and therefore, does not appear to be a true myosin motor. Notably, class-18A myosins and class II myosins copolymerize in vitro and in vivo into bipolar filaments via their extended coiled-coil domains, suggesting a potential role for myosin 18A in the regulation of filament turnover or as an adaptors to link the filaments to different cellular structures or signaling molecules without interfering with NMII motor activity. Alternative splicing of the mammalian myosin 18A gene results in at least 2 isoforms (myosin 18A&#61537; and &#61538;). Both myosin 18A&#61537; and myosin 18A&#61538;&#61472;consist of a motor domain followed by a short neck region, an extended coiled-coil domain, and a C-terminal non-helical tailpiece harboring binding sites for SH3 and PDZ domain-containing proteins. Myosin 18A&#61537; has an N-terminal extension that contains a KE-rich region, an ATP-insensitive actin-binding domain, and a PDZ domain. Knockout of myosin 18A results in embryonic lethality in both mice and flies, suggesting a fundamental role in development. Myosin 18A appears ubiquitously expressed across mammalian tissues with elevated expression and isoform-specific expression in certain cell types. The goal of these studies was to investigate M18A in epithelia-derived generic cells and epithelial tissues. We analyzed the localization of myosin 18A in both polarized MDCK cell sheets and in cryo-sections of various mouse epithelia-containing tissues using a myosin 18A-specific antibody. We show preferential localization of myosin 18A to cell-cell junctions at the apical surface of polarized MDCK cells in culture, where NMII is known to be critical for maintaining epithelial integrity. In tissue sections, such as kidney and intestine, myosin 18A is enriched in proximal tubules and microvilli. Both tissues are also expressing NMII. Additionally, in secretory tissues, such as the pancreas and salivary gland, M18A localizes to the outer surface of secretory granules immediately prior to their secretion. This is similar to the localization kinetics of NMII on these structures, where NMII is known to be essential for maintaining proper hydrostatic pressure for secretion to occur. Preliminary experiments in the salivary gland suggest that M18A might be recruited to granules together with NMII. Together, these data argue that M18A may be regulating NMII as it functions to maintain classic epithelia integrity and as it functions in more specialized processes, such as pancreatic and salivary secretion.